Revista Brasileira de Zootecnia © 2012 Sociedade Brasileira de Zootecnia ISSN 1806-9290 www.sbz.org.br
R. Bras. Zootec., v.41, n.8, p.1890-1898, 2012
In situ and in vitro degradation kinetics and prediction of the digestible neutral detergent fiber of agricultural and agro-industrial byproducts José Augusto Gomes Azevêdo1, Sebastião de Campos Valadares Filho2,3, Edenio Detmann2, Douglas dos Santos Pina4, Mário Fonseca Paulino2, Rilene Ferreira Diniz Valadares5, Luiz Gustavo Ribeiro Pereira6, Jéssika Carolina Moutinho Lima7 1
DCAA/Universidade Estadual de Santa Cruz. Membro do INCT em Ciência Animal. DZO/Universidade Federal de Viçosa. Pesquisador do CNPq. 3 Coordenador do INCT em Ciência Animal. 4 DZO/Universidade Federal de Mato Grosso. 5 DVT/Universidade Federal de Viçosa. Pesquisadora do CNPq. 6 Embrapa Gado de Leite. 7 DZO/Universidade Federal de Viçosa. 2
ABSTRACT - The objective of this study was to evaluate the in situ and in vitro degradation kinetics and to predict the digestible neutral detergent fiber (dNDF) from the incubation times; in situ and in vitro degradation kinetic parameters; and equations fitted for agricultural and agro-industrial byproducts. Byproducts from pineapple, cocoa, palm kernel, corn gluten meal, common bean, sunflower, guava, cassava bark, cassava stems, cassava foliage, papaya, mango, passion fruit and turnips were evaluated. There were differences between the byproducts as for the potentially neutral detergent fiber (NDF) fraction and the in situ NDF degradation rate in the final volume of the gases generated by fibrous carbohydrates (FC), for the lag time and for the in vitro fractional degradation rate of the FC. There was equivalence between the dNDF values predicted in situ and those observed in vivo; however, there was low precision of estimates. The degradability in the in vitro incubation times of 30 and 48 hours presented equivalence with the values observed, but also did not present precision in the estimates. The equations fitted without lignin were not precise and accurate to estimate the dNDF of agricultural and agro-industrial byproducts. The equation with lignin and with the digestion rate obtained by the in vitro method presented more precise estimates. Byproducts from common bean, cassava bark and papaya presented greater NDF availability, whereas those of guava had the lowest NDF availability. The digestible NDF fraction was best predicted with the in situ incubation time of 72 hours. The equation fitted utilizing in vitro or in situ digestion rates enables the prediction of the NDF availability of agricultural and agroindustrial byproducts. Key Words: fibrous carbohydrates, gas production, residues, rumen degradability
Introduction Before utilizing alternative feedstuffs in diets for livestock, it is important to know about their chemical composition, the availability of their nutrients, the behavior in the digestive tract, and to evaluate these feedstuffs in diets for these animals. Among the different components of the feeds for ruminants, the fibrous fraction is of utmost importance in tropical production systems, for it supplies significant amount of energy at a low cost (Detmann et al., 2004). For Kendall et al. (2009), maximizing the ingestion of digestible carbohydrates is important, once the energy necessary for maintenance and production often exceeds the ingestion consumption capacity of high-production animals.
Received April 11, 2004 and accepted March 22, 2012. Corresponding author:
[email protected]
The neutral detergent fiber (NDF) is the main component of the feedstuff that affects the dry matter intake (DMI) of animals of high production (Waldo, 1986). For Oliveira et al. (2011), the following are expected in diets with a high concentration of NDF: reduction of the potentially digestible fraction of the dry matter (DM); increase in the selective ruminal retention as a natural mechanism of compensation and increase in the probability of NDF digestion. However, the NDF is not a homogeneous component. Therefore, feedstuffs with high NDF degradation rate are positively correlated with DMI (Van Soest, 1994). Nevertheless, this does not apply to the NDF content, i.e., feedstuffs with concentration similar to the NDF may have different DMI levels, which is limited by the amount of rumen-undegraded NDF (Andriguetto et al., 1993).
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The digestibility of NDF is dependent on the time it remains inside the digestive tract for hydrolysis and, consequently, its use is affected both by digestion and passage (retention time) rates. The NDF rumen digestibility of the feedstuffs can vary from 0.25 to 0.75 kg/kg for different types of forage (NRC, 2001). The summative equations to predict the digestible fractions of the feedstuffs were developed considering the relations between cause and effect of chemical components and their digestibility in the rumen tract (Van Soest, 1967). However, the biological methods are usually utilized to better characterize the digestible fibrous fraction of the feedstuff (Weiss, 1998). There are doubts as for the exactness of the digestible neutral detergent fiber (dNDF) estimated from the dNDF in vitro (Weiss & Wyatt, 2002). According to Kendall et al. (2009), differences of 5.5 and 8.5% have been observed between the values obtained in vivo and those estimated in vitro during 48 h of incubation for diets with 280 and 320 g/kg NDF, respectively. Likewise, Oba & Allen (2000) observed differences of 9.4 percentage units between the dNDF obtained in vitro during 30 hours of incubation and those observed in vivo with cattle receiving diets based on corn silage. Magalhães (2007) and Silva et al. (2007) observed that the incubation time of 72 hours was the best time for correlation between dNDF in vivo and the degradation of this fraction in situ for tropical forages. Thus, the objective of this study was to evaluate the in situ degradation kinetics, the in vitro gas production kinetics and to predict the dNDF from the incubation times, parameters of in situ and in vivo degradation kinetics and
equations fitted for agricultural and agro-industrial byproducts.
Material and Methods Byproducts from pineapple, cocoa, palm kernel, corn gluten meal, common bean, sunflower, guava, cassava bark, cassava stems, cassava foliage, papaya, mango, passion fruit and turnip were evaluated (Table 1). Byproducts from pineapple (Ananas comosus), guava (Psidium guajava), papaya (Carica papaya), mango (Mangifera indica) and passion fruit (Passiflora ligularis originated from the processing for production of fruit juice, so they were comprised of peel, seeds, the fibrous part retained in the strainers and fruit unsuitable for processing. The byproduct from cocoa was basically composed of the integument which surrounds the seed (nut) after its industrial processing, but also contained little pieces of seeds. The byproduct cassava hulls was composed of strain, tip, bark and inner bark of its root resulting from the pre-cleaning for fabrication of cassava flour and was dried in industrial dryer at 60 ºC. The cassava stem was composed only of the stalk without leaves and was dried in industrial dryer at 60 ºC. The byproduct corn gluten meal was composed of the part of the external membrane of the corn grain which remains after the extraction of the biggest part of the starch, of the gluten and of the germ by the process employed in the production of starch or syrup via wet process. All the byproducts collected were subjected to predrying at 60 ºC for 72 hours and ground in knife mill with 1mm pore diameter, for subsequent analysis of DM, crude protein (CP), organic matter (OM), ether extract (EE) and acid
Table 1 - Chemical composition of 14 agricultural and agro-industrial byproducts Item Pineapple Cocoa Palm kernel Corn gluten meal Common bean Sunflower Guava Papaya Cassava hulls Cassava stem Cassava foliage Mango Passion fruit Turnip
Dry matter1
Organic matter2
139.1 893.4 924.4 857.6 872.2 927.3 285.6 100.7 255.6 899.8 218.5 345.0 195.3 916.2
952.7 925.8 970.4 942.2 955.4 948.7 986.1 949.3 966.3 962.6 918.1 976.1 963.2 942.9
Crude NDIP 3 protein 2 70.9 143.3 161.0 218.6 239.2 368.8 86.2 148.0 37.3 54.4 241.7 50.5 99.7 275.9
504.9 551.4 949.1 382.4 358.3 118.0 267.7 868.7 588.2 471.5 598.0 791.7 179.8 199.3
ADIP 3 443.8 474.6 583.2 132.6 254.4 55.7 188.5 274.1 74.1 213.4 559.6 226.4 77.0 187.3
Ether NDFap 2 extract 2 7.8 50.7 107.1 28.4 19.5 21.3 76.8 72.8 05.9 9.2 23.8 40.0 122.0 242.8
602.0 369.7 523.0 226.8 294.8 186.3 729.6 293.8 157.5 651.1 404.4 325.5 547.7 227.2
NFC 2
ADF 2
272.0 362.2 179.3 468.4 401.9 372.2 93.5 434.8 765.6 247.9 248.2 560.1 193.8 196.9
341.1 400.9 359.4 124.6 66.9 133.1 597.4 327.4 155.6 571.2 362.6 237.8 427.0 196.9
Lignin 2 iNDF 2 iADF 2 37.1 185.4 111.8 14.4 01.8 52.4 221.0 77.4 55.4 200.1 96.1 72.5 77.9 75.3
149.4 321.3 252.9 40.0 4.9 95.0 657.0 78.7 141.6 646.2 339.9 199.7 336.7 179.1
70.2 298.4 162.2 10.4 0.0 76.1 508.8 52.7 110.7 529.3 252.7 147.5 261.4 125.1
NDIP - neutral detergent insoluble protein; ADIP - acid detergent insoluble protein; NDFap - neutral detergent fiber corrected for ash and protein; NFC - non-fibrous carbohydrates; ADF - acid detergent fiber; iNDF - indigestible neutral detergent fiber; iADF - indigestible acid detergent fiber. 1 g/kg of natural matter. 2 g/kg of dry matter. 3 g/kg of crude protein.
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detergent fiber (ADF), according to the methods of the AOAC (1990). In the NDF analyses, samples were treated with thermostable alpha-amylase, without the use of sodium sulfite and corrected for residual ash (Mertens, 1992). The correction of NDF and ADF for the nitrogenous compounds and the estimation of neutral (NDIN) and acid detergent insoluble nitrogen (ADIN) compounds were performed according to Licitra et al. (1996). The lignin contents were obtained by means of solubilization of cellulose by sulfur acid (Van Soest & Robertson, 1985). The contents of non-fibrous carbohydrates (NFC) of the byproducts, expressed in g/kg DM, were calculated according to Hall (2000) as 100- (g/kgNDF + g/kgCP + g/ kgEE + g/kgMM) and total digestible nutrients (TDN) were calculated as: TDN = g/kg digestible CP + g/kg digestible NDF + g/kg digestible NFC + 2.25*g/kg digestible EE. For in situ incubations, samples were dried in oven at 60 ºC for 72 hours, processed in knife mill with 2 mm sieve and homogenized, forming a composite sample for each residue for further incubation. For the evaluation of indigestible NDF (iNDF) and indigestible ADF (iADF) contents, three replicates of each byproduct were conditioned in non-woven fabric bags of 100 g/m2 measuring 4 × 5 cm and following the ratio of 20 mg DM/cm2 surface suggested by Nocek (1988) and incubation time of 264 hours, proposed by Casali et al. (2008). For incubations, three crossbred (Holstein × Zebu) rumen-cannulated castrated cattle of average weight of 260 kg were utilized. Animals received a diet containing 700 g/kg roughage and 300 g/kg concentrate. Byproducts were incubated (2 g DM in each bag) in duplicate, in non-woven textile bags of 100 g/m2, following the ratio of 20 gm DM/cm2 suggested by Nocek (1988), in the following incubation times, in descending order: 144, 120, 96, 72, 48, 24, 12, 6 and 3 h. After removed from the rumen, along with time zero, they were taken to running water until total clearing and then transferred to forcedventilation oven (60 ºC), where they were kept for 72 hours. They were then sequentially dried in non-ventilated oven (105 ºC for 45 minutes), conditioned in dissector and weighed for the obtainment of the non-digested DM. After, bags were treated with neutral detergent fiber (Mertens, 2002) for 60 minutes, in autoclave (Pell & Schofield, 1993) at 105 ºC, washed in hot water, acetone, weighed and dried, according to the aforementioned procedure, for quantification of non-digested NDF. The in situ degradation data were obtained by the difference of weight, found for each component, between the weighings done before and after ruminal incubation and
expressed in percentage. For the estimation of the potentially degradable fraction, the exponential decay model was utilized, corrected for the lag period (L), described by Mertens (1976), according to the formula: ^ = B* exp (-kd*(t - L)) + I, Y ^ in which Y accounts for the non-digested DM or NDF residue in time t (%); B is the potentially degradable fraction of the fiber (%); kd is the rumen degradation dynamics of fraction B (h -1); t is the incubation time, in hours; L is the lag time (h); and I is the undegradable fraction (%), which represents the iNDF contents. The NDF effectively degraded fraction (NDFEDF) was estimated by the equation (Mertens & Loften, 1980): NDFEDF = B*kd*exp(-kp*L)/(kd + kp), in which kd accounts for the passage rate of the digesta through the rumen, assuming kp value equal to 0.02 h-1. For the in vitro incubations, calibrated syringes were utilized, according to procedure described by Getchew et al. (2004). Syringes with capacity of 100 mL were previously washed with distilled water, dried in oven and subsequently lubricated with Vaseline, in which approximately 200 mg of the byproduct studied were placed. The macro and mineral buffer solution, described by Menke & Steingass (1988), was prepared prior to incubation and kept heated at 39 ºC, under continuous gasification by CO 2, on a stirrer. The animals which donated the inoculate were the same of the in situ experiment. The ruminal fluid was taken manually in the morning, before the supply of diet, from several parts of the rumen, and stored in previously heated (39 ºC) thermos bottles and immediately taken to laboratory. In an acclimatized room of the lab, (39 ºC), the rumen fluid was filtered, passing through two layers of cotton gauze. After, the rumen fluid was added to the buffer solution at a 2:1 ratio (v/v), under continuous CO 2 injection. Three syringes were utilized per feedstuff, in which 30 mL of the buffered ruminal fluid were added to the substrate. After closure of the tip of the syringe with a clip attached to a silicone rubber to impede sample leaking, the piston was introduced until there was total removal of gases, and then the syringe was gently agitated. With the syringe tip closed, the initial volume was recorded. At every two hours, reading was performed through records of gases volume, and the syringe was gently agitated. The incubation times utilized were 0, 2, 4, 6, 8, 10, 12, 24, 26, 28, 30, 32, 36, 48, 52, 54, 56, 60 and 72 hours. The results were corrected for blank (syringe containing buffered ruminal fluid, without sample) and for the standard (tifton 85 grass hay), at 24 h of incubation. R. Bras. Zootec., v.41, n.8, p.1890-1898, 2012
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The kinetic variables of fibrous carbohydrates (FC) and NFC were estimated by the technique of in vitro gas production. Bicompartimental model was used, fitted to the curves of cumulative gas production (Schofield et al., 1994): V = VFNFC / (1 + exp (2 - 4*kdNFC*(T - L))) + VFFC / (1 + exp(2 - 4*kdFC*(T - L))), in which: VF NFC is equivalent to the maximum volume of gases from the NFC fraction; kd NFC is the degradation rate (h -1 ) of this fraction (NFC); VF FC is the maximum volume of the gases from the FC fraction; kdFC is the degradation rate (h-1) of FC; and T and L are the incubation (hours) and lag (hours) times, respectively. After estimation of the kinetic variables of production of gases from carbohydrates, the degradation curves of FC in function of incubation time were constructed, for the data obtained by the method of gas production. For incubations, glass flasks were utilized according to procedure described by Schofield et al. (1994). Flaks with capacity of 50 mL were previously washed with distilled water, dried in oven and had approximately 200 mg of the byproduct studied inserted in them. Macro and micromineral buffer solution, in addition to the ruminal fluid, was the same as described in the experiment for kinetics of in vitro gas production. Three flasks were utilized per byproduct; every one of them had 30 mL of the buffered ruminal liquid added to the substrates and were immediately sealed with rubber corks and aluminum rings aiming to ensure complete maintenance of the gases inside them. After 30 and 48 hours of incubation, flasks were removed from the acclimatized room and taken to refrigerator for 4 ºC, for ceasing the fermentative process. After, 30 mL of neutral detergent solution were added to each flask (Mertens, 2002) and then taken to be autoclaved for 60 minutes, at 105 ºC, according to technique proposed by Pell & Schofield (1993). Next, the contents of each glass flasks were filtered in filter crucible of zero-porosity, washed with hot distilled water and acetone and dried in oven at 105 ºC for 16 hours. For the prediction of the NDF digestible fiber, the equation proposed by Conrad et al. (1984), adapted by Tedeschi et al. (2009) and a modification of the equation adapted by Tedeschi et al. (2009) were evaluated, resulting in the following equations: - Equation proposed by Conrad et al. (1984) and adapted by Tedeschi et al. (2009):
- Modified equation:
In this evaluation, passage rate (Kp) of 0.02 h-1 and two digestion rates (Kd) were considered (obtained in vitro by the technique of gas production and in situ). The results obtained by Azevêdo (2009) were utilized for the in vivo dNDF information of agricultural and agroindustrial byproducts (Table 2). For the procedures of validation of the digestible fractions observed and predicted by the incubation times, model parameters of the fitted equations were based on the fitting of the simple linear regression models, and estimates of the regression parameters tested by the joint null hypothesis according to Mayer et al. (1994): Ho: β0 = 0 and β1 = 1 X Ha: non-Ho. In the case of non-rejection of the null hypothesis, it is concluded that there is equivalence between the observed and predicted values. The mean bias (MB) was calculated (Cochran & Cox, 1957) according to the following equation: ; in which: x = observed values; y = predicted values. The concordance correlation coefficient (CCC), also known as reproducibility index, which considers exactness and precision simultaneously, was calculated according to Lin (1989). The comparative evaluation of the prediction efficiency was performed by the evaluation of the mean square prediction error (MSPE), as described by Bibby & Toutenburg (1977), following the equation below: , in which: x = observed values; y = predicted values. It is necessary to stress that for all the variance calculations, the total observations (n) was utilized as divisor.
Table 2 - Digestible neutral detergent fiber (dNDF) in different agricultural and agro-industrial byproducts1 Agricultural and agro-industrial byproducts Pineapple Cocoa Palm kernel Corn gluten meal Common bean Sunflower Guava Papaya Cassava hulls Cassava stem Cassava foliage Mango Passion fruit Turnip 1
dNDF g/kg of DM 461.1 64.9 333.1 128.5 209.8 120.2 145.6 201.3 55.6 434.2 350.7 221.2 258.6 105.2
Information obtained in vivo by Azevêdo (2009).
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The data on the NDF degradability parameters and production of gases from NFC and FC obtained in the different methods (in situ and in vitro) and incubation times were fitted by non-linear regression through the GaussNewton method, according to the respective models previously informed. Variance analyses were conducted, by applying the F test. For the variables whose F test was significant, the means were compared utilizing the Scott Knott criterion. For all statistical procedures, the critical level for probability of type I error was fixed at 0.05. All statistical procedures were performed with software SAS (Statistical Analysis System, version 9.0) and MES (Model Evaluation System, version 3.0.11).
The values estimated for the in situ NDF degradability indicate that the byproducts from pineapple, palm kernel, corn gluten meal, common bean, sunflower, papaya and mango showed greater (P
